(1) Field of the Invention
This invention relates to a method of controlling cleavage of a polypeptide by OmpT protease using novel cleavage and recognition sites which have been found by examining the substrate specificity of Escherichia coli OmpT protease.
In one aspect, the present invention relates to a method of cleaving polypeptides by using OmpT protease. More particularly, it relates to a method of cleaving polypeptides by utilizing novel cleavage and recognition sites of OmpT protease.
In another aspect, the present invention relates to a method of excising physiologically active peptides, proteins and derivatives thereof from fusion proteins with the use of OmpT protease. More particularly, the present inventors examined the substrate specificity of OmpT protease to find a novel cleavage method, cleavage sites and recognition sites. Thus, one embodiment of the invention relates to a method of efficiently producing physiologically active peptides, a protein and derivatives thereof from fusion proteins by using the properties of the novel cleavage sites and recognition sites.
The present invention further relates to a method of avoiding cleavage of polypeptides by OmpT protease at undesired sites. In particular, it relates to a method of avoiding cleavage of physiologically active peptides, proteins or derivatives thereof by OmpT protease produced by host cells. Thus, the present invention provides a method of making the physiologically active peptides, proteins or derivatives thereof not (or hardly) cleavable by OmpT protease by converting the amino acid sequences at the OmpT protease cleavage sites or in the vicinity thereof.
(2) Description of the Related Art
E. coli OmpT protease is an E. coli outer membrane protease that selectively cleaves mainly bonds between basic amino acid pairs (Sugimura, K. and Nishihara, T. J. Bacteriol. 170: 5625-5632, 1988). This enzyme has a molecular weight of 36,000 and seemingly falls within the category of serine proteases. Sugimura et al. examined the substrate specificity of this OmpT protease and reported that the enzyme specifically cleaves the peptide bonds at the center of basic amino acid pairs of arginine-arginine, lysine-lysine, arginine-lysine and lysine-arginine. In addition, cleavage sites in amino acid sequences other than the above pairs has been found. Namely, it has been reported that cleavage by OmpT protease arises at arginine-methionine (Zhao, G-P. and Somerville, R. L. J. Biol. Chem. 268, 14912-14920, 1993), arginine-alanine (Lassen, S. F. et al. Biochem. Int. 27: 601-611, 1992) and arginine-valine (Maurer, J. J. Bacteriol. 179: 794-804, 1997). This protease is characterized in that it cleaves not all proteins and peptides containing these sequences but exclusively cleaves specific proteins and peptides at specific sites. For example, γ-interferon contains the sequences described above at 11 sites but only 2 sites among them are exclusively cleavable by OmpT protease (Sugimura, K. and Higashi, N.J. Bacteriol. 170: 3650-3654, 1988). T7 RNA polymerase contains the sequences described above at 17 sites but only 2 sites among them are exclusively cleavable therewith (Grodberg, J. and Dunn, J. J. J. Bacteriol. 170: 1245-1253, 1988). The above-described data indicates that the known information on the cleavage sites of OmpT protease is not applicable to estimation of cleavage sites of OmpT protease. That is, OmpT protease differs from enzymes such as AP-1 and trypsin which are commonly employed in peptide mapping of proteins, in that the cleavage site of AP-1 and trypsin can be estimated on the basis of known data. Since the cleavage by OmpT protease arises at specific sites of proteins or peptides, it is anticipated that amino acid sequences other than the amino acid sequences as described above (namely, the N-terminal and C-terminal amino acid sequences of the cleavage site) may participate in the cleavage. However, it still remains unknown so far what amino acid sequence allows (or does not allow) the cleavage.
However, OmpT protease has found use in excising target polypeptides from fusion proteins constructed by gene recombination techniques, since it has high specificity to cleavage sites and is one of endogenous proteases of E. coli. In order to produce cholesterol esterase by using E. coli, Hanke et al. prepared a fusion protein consisting of the esterase and E. coli hemolysin A protein. The fusion protein was secreted in culture supernatant, and then cleaved by the outer membrane protease OmpT. Consequently, they could obtain active cholesterol esterase successfully. Hanke et al. employed a linker having an arginine-lysine sequence and cleaved this sequence by OmpT protease (Hanke, C et. al. Mol. Gen. Genet. 233: 42-48, 1992).
The present inventors found that OmpT protease is resistant to denaturing agents and that fusion proteins expressed as inclusion bodies can be cleaved in the presence of a denaturing agent by taking advantage of the above property. Namely, the present inventors successfully produced a V8 protease derivative having enzymatic activity by expressing an S. aureus V8 protease derivative fusion protein as an inclusion body in an E. coli expression system, solubilizing the same by urea, releasing the V8 protease derivative moiety from the fusion protein by using OmpT protease in the presence of urea and finally refolding (Yabuta, M., Ochi, N. and Ohsuye, K. Appl. Microbiol. Biotechnol. 44:118-125, 1995).
To release target peptides or target proteins from fusion proteins, it has been a practice to employ enzymes having high specificity to amino acid sequences. Known examples of proteases employed for this purpose include Xa factor, thrombin, enterokinase and the like which are enzymes originating in mammals and are supplied only in a small amount at a high cost. Therefore, these enzymes are unsuitable for the industrial treatment of peptides and proteins by the fusion protein method on a mass scale. When the target peptide or protein is to be used as a medicine, it is also required to take into consideration viral contamination originating in the enzymes. In contrast thereto, OmpT protease is clearly superior to these enzymes in supply, cost and safety because of its origination in E. coli. 
However, the substrate specificity of this protease has not been sufficiently studied yet and, therefore, it is difficult at the present stage to arbitrarily design the cleavage site at the desired part to be excised. Moreover, OmpT protease cleaves not all of the sequences reported above (i.e., arginine-arginine, lysine-lysine, arginine-lysine, lysine-arginine, arginine-methionine, arginine-alanine and arginine-valine) but exclusively specific sites in proteins. When one of sequences consisting of these two amino acids is merely located in a linker site of fusion proteins, therefore, this site cannot always be cleaved by OmpT protease. Even though it can be cleaved, OmpT protease cleaves the peptide bond at the center of the cleavage site consisting of two amino acids. Therefore, the amino acid located at the +1-position of the cleavage site will remain as the N-terminus of the target polypeptide when this enzyme releases the target polypeptide from a fusion protein consisting of a protective peptide, a linker peptide including the OmpT protease cleavage site, and a target polypeptide in this order. Moreover, this added amino acid cannot be arbitrarily selected but restricted to arginine, lysine, valine, alanine or methionine on the basis of the recognition sequences of OmpT protease reported hitherto. These properties of OmpT protease are unfavorable as a protease to be used in cleaving fusion proteins.
On the other hand, it is known that the cleavage efficiency of papain, which is a protease, is affected not only by the sequence of the cleavage site in the substrate but also by the amino acid sequences in the vicinity thereof (Schechter, I. and Berger, A. Biochem. Biophys. Res. Commun. 27: 157-162 1967). Recently, detailed studies have also been made on Kex2 (Rockwell, N. C., Wang, G. T., Krafft, G. A. and Fuller, R. S. Biochemistry 36, 1912-1917 1997) and furin (Krysan, D. J., Rockwell, N. C. and Fuller, R. S. J. Biol. Chem. 274, 23229-23234 1999) which are proteases cleaving the C-terminal side of basic amino acid pairs. In Kex2 and furin, the consensus sequences at the cleavage sites and amino acid sequences in the vicinity thereof have been clarified by comparing the amino acid sequences of the substrates. In the case of OmpT protease, it is considered, on the basis of the comparison of the substrates known hitherto, that arginine or lysine is essentially required as the amino acid at the 1-position in the N-terminal side of the cleavage site but no other clear consensus sequence has been found out so far. Although it is presumed that the recognition of the cleavage site and the cleavage efficiency of OmpT protease might be also affected not only by the cleavage site in the substrate but also by the amino acid sequences in the vicinity thereof, it is impossible at the present stage to control the cleavage by OmpT protease by using these properties.
In the present invention, the location of each amino acid in a given polypeptide is represented as follows. A sequence site consisting of two arbitrary consecutive amino acids in the polypeptide is referred to as the cleavage site or the site to be cleaved by OmpT protease. Between the amino acids concerning this site, the amino acid in the N-terminal side is referred to as the −1-position while the amino acid in the C-terminal side is referred to as the +1-position. The amino acids at the 1st, 2nd, 3rd, and so on in the N-terminal side of this site are referred to respectively as the amino acids at the −1-, −2-, −3-positions and so on, while the amino acids at the 1st, 2nd, 3rd, and so on in the C-terminal side of this site are referred to respectively as the amino acids at the +1-, +2-, +3-positions and so on. When an amino acid substitution is introduced into this site or in the vicinity thereof so that the site becomes not cleavable or cleavable, the corresponding amino acids in the sequence are represented by the above-described numbering.
For example, when the amino acid sequence leucine-tyrosine-lysine-arginine-histidine is to be cleaved at the bond between lysine and arginine (i.e., the two arbitrary consecutive amino acids), leucine, tyrosine, lysine, arginine and histidine serve respectively as the amino acids at the −3-, −2-, −1-, +1- and +2-positions.